Mario de Oliveira Neto

Biomass-to-bio-products application of feruloyl esterase from Aspergillus clavatus

The structural polysaccharides contained in plant cell walls have been pointed to as a promising renewable alternative to petroleum and natural gas. Ferulic acid is a ubiquitous component of plant polysaccharides, which is found in either monomeric or dimeric forms and is covalently linked to arabinosyl residues. Ferulic acid has several commercial applications in food and pharmaceutical industries. The study herein introduces a novel feruloyl esterase from Aspergillus clavatus (AcFAE). Along with a comprehensive functional and biophysical characterization, the low-resolution structure of this enzyme was also determined by small-angle X-ray scattering. In addition, we described the production of phenolic compounds with antioxidant capacity from wheat arabinoxylan and sugarcane bagasse using AcFAE. The ability to specifically cleave ester linkages in hemicellulose is useful in several biotechnological applications, including improved accessibility to lignocellulosic enzymes for biofuel production.

Modular Hyperthermostable Bacterial Endo-β-1,4-Mannanase: Molecular Shape, Flexibility and Temperature-Dependent Conformational Changes

Endo-β-1,4-mannanase from Thermotoga petrophila (TpMan) is a hyperthermostable enzyme that catalyzes the hydrolysis of β-1,4-mannoside linkages in various mannan-containing polysaccharides. A recent study reported that TpMan is composed of a GH5 catalytic domain joined by a linker to a carbohydrate-binding domain. However, at this moment, there is no three-dimensional structure determined for TpMan. Little is known about the conformation of the TpMan as well as the role of the length and flexibility of the linker on the spatial arrangement of the constitutive domains. In this study, we report the first structural characterization of the entire TpMan by small-angle X-ray scattering combined with the three-dimensional structures of the individual domains in order to shed light on the low-resolution model, overall dimensions, and flexibility of this modular enzyme at different temperatures. The results are consistent with a linker with a compact structure and that occupies a small volume with respect to its large number of amino acids. Furthermore, at 20°C the results are consistent with a model where TpMan is a molecule composed of three distinct domains and that presents some level of molecular flexibility in solution. Even though the full enzyme has some degree of molecular flexibility, there might be a preferable conformation, which could be described by the rigid-body modeling procedure. Finally, the results indicate that TpMan undergoes a temperature-driven transition between conformational states without a significant disruption of its secondary structure. Our results suggest that the linker can optimize the geometry between the other two domains with respect to the substrate at high temperatures. These studies should provide a useful basis for future biophysical studies of entire TpMan.

Development and Biotechnological Application of a Novel Endoxylanase Family GH10 Identified from Sugarcane Soil Metagenome

Metagenomics has been widely employed for discovery of new enzymes and pathways to conversion of lignocellulosic biomass to fuels and chemicals. In this context, the present study reports the isolation, recombinant expression, biochemical and structural characterization of a novel endoxylanase family GH10 (SCXyl) identified from sugarcane soil metagenome. The recombinant SCXyl was highly active against xylan from beechwood and showed optimal enzyme activity at pH 6,0 and 45°C. The crystal structure was solved at 2.75 Å resolution, revealing the classical (β/α)8-barrel fold with a conserved active-site pocket and an inherent flexibility of the Trp281-Arg291 loop that can adopt distinct conformational states depending on substrate binding. The capillary electrophoresis analysis of degradation products evidenced that the enzyme displays unusual capacity to degrade small xylooligosaccharides, such as xylotriose, which is consistent to the hydrophobic contacts at the +1 subsite and low-binding energies of subsites that are distant from the site of hydrolysis. The main reaction products from xylan polymers and phosphoric acid-pretreated sugarcane bagasse (PASB) were xylooligosaccharides, but, after a longer incubation time, xylobiose and xylose were also formed. Moreover, the use of SCXyl as pre-treatment step of PASB, prior to the addition of commercial cellulolytic cocktail, significantly enhanced the saccharification process. All these characteristics demonstrate the advantageous application of this enzyme in several biotechnological processes in food and feed industry and also in the enzymatic pretreatment of biomass for feedstock and ethanol production.

Effects of autohydrolysis of Eucalyptus urograndis and Eucalyptus grandis on influence of chemical components and crystallinity index.

Samples of Eucalyptus urograndis and Eucalyptus grandis sawdust were autohydrolyzed in aqueous conditions to reach temperatures in the range 110-190°C and reaction times of 0-150min in a minireactor. In each minireactor were used a liquor:wood ratio (10:1 L:kg dry wood), in order to assess the effects of the autohydrolysis severity and the crystalline properties of cellulose. The content of extractives, lignin, holocellulose, cellulose, hemicelluloses and crystallinity index obtained from the solid fraction after autohydrolysis of sawdust were determined. This study demonstrated that the hemicelluloses were extensively removed at 170 and 190°C, whereas cellulose was partly degraded to Eucalyptus urograndis and Eucalyptus grandis sawdust. The lignin content decreased, while the extractives content increased. It was defined that during autohydrolysis, had a slight decreased on crystalline structure of cellulose of Eucalyptus urogandis and Eucalyptus grandis.

Conformational changes in a hyperthermostable glycoside hydrolase: enzymatic activity is a consequence of the loop dynamics and protonation balance.

Endo-β-1, 4-mannanase from Thermotoga petrophila (TpMan) is a modular hyperthermostable enzyme involved in the degradation of mannan-containing polysaccharides. The degradation of these polysaccharides represents a key step for several industrial applications. Here, as part of a continuing investigation of TpMan, the region corresponding to the GH5 domain (TpManGH5) was characterized as a function of pH and temperature. The results indicated that the enzymatic activity of the TpManGH5 is pH-dependent, with its optimum activity occurring at pH 6. At pH 8, the studies demonstrated that TpManGH5 is a molecule with a nearly spherical tightly packed core displaying negligible flexibility in solution, and with size and shape very similar to crystal structure. However, TpManGH5 experiences an increase in radius of gyration in acidic conditions suggesting expansion of the molecule. Furthermore, at acidic pH values, TpManGH5 showed a less globular shape, probably due to a loop region slightly more expanded and flexible in solution (residues Y88 to A105). In addition, molecular dynamics simulations indicated that conformational changes caused by pH variation did not change the core of the TpManGH5, which means that only the above mentioned loop region presents high degree of fluctuations. The results also suggested that conformational changes of the loop region may facilitate polysaccharide and enzyme interaction. Finally, at pH 6 the results indicated that TpManGH5 is slightly more flexible at 65°C when compared to the same enzyme at 20°C. The biophysical characterization presented here is well correlated with the enzymatic activity and provide new insight into the structural basis for the temperature and pH-dependent activity of the TpManGH5. Also, the data suggest a loop region that provides a starting point for a rational design of biotechnological desired features.

Insights on the structure of native CNF, an endogenous phospholipase A2 inhibitor from Crotalus durissus terrificus, the South American rattlesnake

Several snake species possess endogenous phospholipase A2 inhibitors (sbPLIs) in their blood plasma, the primary role of which is protection against an eventual presence of toxic phospholipase A2 (PLA2) from their venom glands in the circulation. These inhibitors have an oligomeric structure of, at least, three subunits and have been categorized into three classes (α, β and γ) based on their structural features. SbγPLIs have been further subdivided into two subclasses according to their hetero or homomeric nature, respectively. Despite the considerable number of sbγPLIs described, their structures and mechanisms of action are still not fully understood. In the present study, we focused on the native structure of CNF, a homomeric sbγPLI from Crotalus durissus terrificus, the South American rattlesnake. Based on the results of different biochemical and biophysical experiments, we concluded that, while the native inhibitor occurs as a mixture of oligomers, tetrameric arrangement appears to be the predominant quaternary structure. The inhibitory activity of CNF is most likely associated with this oligomeric conformation. In addition, we suggest that the CNF tetramer has a spherical shape and that tyrosinyl residues could play an important role in the oligomerization. The carbohydrate moiety, which is present in most sbγPLIs, is not essential for the inhibitory activity, oligomerization or complex formation of the CNF with the target PLA2. A minor component, comprising no more than 16% of the sample, was identified in the CNF preparations. The amino-terminal sequence of that component is similar to the B subunits of the heteromeric sbγPLIs; however, the role played by such molecule in the functionality of the CNF, if any, remains to be determined.

Non-productive adsorption of bacterial β-glucosidases on lignins is electrostatically modulated and depends on the presence of fibronection type III-like domain

Non-productive adsorption of cellulases onto lignins is an important mechanism that negatively affects the enzymatic hydrolysis of lignocellulose biomass. Here, we examined the non-productive adsorption of two bacterial β-glucosidases (GH1 and GH3) on lignins. The results showed that β-glucosidases can adsorb to lignins through different mechanisms. GH1 β-glucosidase adsorption onto lignins was found to be strongly pH-dependent, suggesting that the adsorption is electrostatically modulated. For GH3 β-glucosidase, the results suggested that the fibronectin type III-like domain interacts with lignins through electrostatic and hydrophobic interactions that can partially, or completely, overcome repulsive electrostatic forces between the catalytic domain and lignins. Finally, the increase of temperature did not result in the increase of β-glucosidases adsorption, probably because there is no significant increase in hydrophobic regions in the β-glucosidases structures. The data provided here can be useful for biotechnological applications, especially in the field of plant structural polysaccharides conversion into bioenergy and bioproducts.

Biochemical and biophysical properties of a metagenome-derived GH5 endoglucanase displaying an unconventional domain architecture

Endoglucanases are key enzymes in the degradation of cellulose, the most abundant polymer on Earth. The aim of this work was to perform the biochemical and biophysical characterization of CelE2, a soil metagenome derived endoglucanase. CelE2 harbors a conserved domain from glycoside hydrolase family 5 (GH5) and a C-terminal domain with identity to Calx-beta domains. The recombinant CelE2 displayed preference for hydrolysis of oat beta-glucan, followed by lichenan and carboxymethyl cellulose. Optimum values of enzymatic activity were observed at 45°C and pH 5.3, and CelE2 exhibited considerable thermal stability at 40°C for up to 360min. Regarding the cleavage pattern on polysaccharides, the release of oligosaccharides with a wide degree of polymerization indicated a characteristic of endoglucanase activity. Furthermore, the analysis of products generated from the cleavage of cellooligosaccharides suggested that CelE2 exhibited transglycosylation activity. Interestingly, the presence of CaCl2 positively affect CelE2, including in the presence of surfactants. SAXS experiments provided key information on the effect of CaCl2 on the stability of CelE2 and dummy atom and rigid-body models were generated. To the best of our knowledge this is the first biochemical and biophysical characterization of an endoglucanase from family GH5 displaying this unconventional modular organization.

Biophysical and biochemical studies of a major endoglucanase secreted by Xanthomonas campestris pv. campestris.

Endoglucanases are the main cellulolytic enzymes secreted by the bacterium Xanthomonas campestris pv. campestris (Xcc). The major endoglucanase exported by this bacterium into an external milieu is an enzyme XccCel5A, which belongs to GH5 family subfamily 1 and is encoded by the gene engXCA. We purified XccCel5A using ammonium sulfate precipitation followed by size exclusion chromatography and identified it by zymogram analysis. Circular dichroism and fluorescence spectroscopy studies showed that XccCel5A is stable in a wide pH range and up to about 55°C and denatures at the higher temperatures. The optimal conditions for enzyme activity were identified as T=45°C and pH=7.0. Under the optimum conditions the catalytic efficiency (kcat/KM) of the enzyme was determined as 5.16×10(4)s(-1)M(-1) using carboxymethylcellulose (CMC) as a substrate. Our SAXS studies revealed extended tadpole-shape molecular assembly, typical for cellulases, and allowed to determine an overall shape of the enzyme and a relative position of the catalytic and cellulose binding domains.

SAXS Studies of the Endoglucanase Cel12A from Gloeophyllum trabeum Show Its Monomeric Structure and Reveal the Influence of Temperature on the Structural Stability of the Enzyme

Endoglucanases are key enzymes applied to the conversion of biomass aiming for second generation biofuel production. In the present study we obtained the small angle X-ray scattering (SAXS) structure of the G. trabeum endo-1,4-β-glucanase Cel12A and investigated the influence of an important parameter, temperature, on both secondary and tertiary structure of the enzyme and its activity. The CD analysis for GtCel12A revealed that changes in the CD spectra starts at 55 °C and the Tm calculated from the experimental CD sigmoid curve using the Boltzmann function was 60.2 ± 0.6 °C. SAXS data showed that GtCel12A forms monomers in solution and has an elongated form with a maximum diameter of 60 ± 5 Å and a gyration radius of 19.4 ± 0.1 Å as calculated from the distance distribution function. Kratky analysis revealed that 60 °C is the critical temperature above which we observed clear indications of denaturation. Our results showed the influence of temperature on the stability and activity of enzymes and revealed novel structural features of GtCel12A.